The invention relates to the discovery and characterization of Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83. Tango-71 is a human protein which is approximately 90% identical to murine ADAMTS-1. Tango-73is a human protein that is 48% identical to rate RVP.1 (Briehl et al., Mol. Endocrinol. 5:1381, 1991). Tango-74 is a human protein with homology to TRAIL receptor (Pan et al., Science 276:111, 1997). Tango-76 is a rat protein which is approximately 40% identical to murine ADAMTS-1. Tango-83 is expressed by stimulated human astrocytes.
The invention features isolated nucleic acid molecules encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptides; isolated nucleic acid molecules encoding polypeptides which are substantially similar to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83; and isolated nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having the sequence of the protein coding portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.
The invention also features a host cell which includes an isolated nucleic acid molecule encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 and a nucleic acid vector (e.g., an expression vector; a vector which includes a regulatory element; a vector which includes a regulatory element selected from the group consisting of the cytomegalovirus hCMV immediate early gene, the early promoter of SV40 adenovirus, the late promoter of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage xcex, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast xcex1-mating factors; vector which includes a regulatory element which directs tissue-specific expression; a vector which includes a reporter gene; a vector which includes a reporter gene selected from the group selected from the group consisting of xcex2-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding xcex2-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT); a vector that is a plasmid, a vector that is a virus; and a vector that is a retrovirus) containing an isolated nucleic acid molecule encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83.
The invention also features substantially pure Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83 polypeptides; a substantially pure polypeptide which includes a first portion and a second portion, the first portion including a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide and the second portion including a detectable marker.
The invention also features an antibody that selectively binds to a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide (e.g., a monoclonal antibody).
The invention also features a pharmaceutical composition which includes a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide.
The invention includes methods for diagnosing a disorder associated with aberrant expression of a protein of the invention (i.e., Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83), the method including obtaining a biological sample from a patient and measuring the expression of the protein in the biological sample, wherein increased or decreased expression of the protein in the biological sample compared to a control indicates that the patient suffers from a disorder associated with aberrant expression of the protein.
The invention encompasses isolated nucleic acid molecules encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 or a polypeptide fragment thereof; vectors containing these nucleic acid molecules; cells harboring recombinant DNA encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83; fusion proteins which include all or a portion of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83; transgenic animals which express Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83; and recombinant knock-out animals which fail to express Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83.
The invention encompasses nucleic acids that have a sequence that is substantially identical to a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 nucleic acid sequence. A nucleic acid molecule which is substantially identical to a given reference nucleic acid molecule is hereby defined as a nucleic acid molecule having a sequence that has at least 85%, preferably 90%, and more preferably 95%, 98%, 99% or more identity to the sequence of the given reference nucleic acid molecule.
The invention also includes polypeptides which are substantially identical to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 (e.g., polypeptides that are substantially identical to the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10).
A polypeptide which is xe2x80x9csubstantially identicalxe2x80x9d to a given reference polypeptide molecule is a polypeptide having an amino acid sequence that has at least 85%, preferably 90%, and more preferably 95%, 98%, 99% or more identity to the amino acid sequence of the given reference polypeptide.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)xc3x97100). Preferably, the two sequences are the same length.
The determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
The nucleic acid molecules of the invention can be inserted into vectors, as described below, which will facilitate expression of the insert. The nucleic acid molecules and the polypeptides they encode can be used directly as diagnostic or therapeutic agents, or (in the case of a polypeptide) can be used to generate antibodies that, in turn, are therapeutically useful. Accordingly, expression vectors containing the nucleic acid molecules of invention, cells transfected with these vectors, the polypeptides expressed, and antibodies generated, against either the entire polypeptide or an antigenic fragment thereof, are among the preferred embodiments.
A transformed cell is any cell into which (or into ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding a polypeptide of the invention (e.g., a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide).
An isolated nucleic acid molecule is a nucleic acid molecule that is separated from the 5xe2x80x2 and 3xe2x80x2 coding sequences with which it is immediately contiguous in the naturally occurring genome of an organism. Isolated nucleic acid molecules include nucleic acid molecule which are not naturally occurring, e.g., nucleic acid molecules created by recombinant DNA techniques.
Nucleic acid molecules include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand.
The invention also encompasses nucleic acid molecules that hybridize, preferably under stringent conditions, to a nucleic acid molecule encoding a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide (e.g., a nucleic acid molecule having the sequence shown in SEQ ID NO:1, 3, 5, 7, or 9). Preferably the hybridizing nucleic acid molecule consists of 400, more preferably 200 nucleotides. Preferred hybridizing nucleic acid molecules have a biological activity possessed by Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83.
The invention also features substantially pure or isolated Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptides, including those that correspond to various functional domains of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83, or fragments thereof.
The polypeptides of the invention can be produced recombinantly, chemically synthesized, or purified from tissues in which they are naturally expressed, according to standard biochemical methods of purification.
Also included in the invention are functional polypeptides, which possess one or more of the biological functions or activities of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. These functions include the ability to bind some or all of the proteins which normally bind to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. A functional polypeptide is also considered within the scope of the invention if it serves as an antigen for production of antibodies that specifically bind to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. In many cases, functional polypeptides retain one or more domains present in the naturally-occurring form of the polypeptide.
The functional polypeptides may contain a primary amino acid sequence that has been modified from those disclosed herein. Preferably these modifications consist of conservative amino acid substitutions, as described herein.
The terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used herein to describe any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). Thus, the term xe2x80x9cTango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptidesxe2x80x9d includes full-length, naturally occurring Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 protein, as well a recombinantly or synthetically produced polypeptide that correspond to a full-length naturally occurring Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 protein or to particular domains or portions of a naturally occurring protein. The term also encompasses mature Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 which has an added amino-terminal methionine (useful for expression in prokaryotic cells).
The term xe2x80x9cpurifiedxe2x80x9d as used herein refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
Polypeptides or other compounds of interest are said to be xe2x80x9csubstantially purexe2x80x9d when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The invention also features antibodies, e.g., monoclonal, polyclonal, and engineered antibodies, which specifically bind Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. An antibody which specifically binds to a given antigen is an antibody that recognizes and binds to a particular antigen, but which does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, which includes Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83.
The invention also features antagonists and agonists of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 that inhibit one or more of the biological activities of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. Suitable antagonists can include small molecules (i.e., molecules with a molecular weight below about 500), large molecules (i.e., molecules with a molecular weight above about 500), neutralizing antibodies, polypeptides which compete with a native form of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 for binding to a protein, and nucleic acid molecules that interfere with transcription of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 (for example, antisense nucleic acid molecules and ribozymes). Agonists of Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83 also include small and large molecules, and antibodies other than neutralizing antibodies.
The invention also features molecules which can increase or decrease the expression of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 (e.g., by influencing transcription or translation). Small molecules (i.e., molecules with a molecular weight below about 500), large molecules (i.e., molecules with a molecular weight above about 500), and nucleic acid molecules that can be used to inhibit the expression of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 (for example, antisense and ribozyme molecules) or to enhance their expression (for example, molecules that bind to a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 transcription regulatory sequences and increase Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 transcription).
In addition, the invention features substantially pure polypeptides that functionally interact with Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83, and the nucleic acid molecules that encode them.
The invention encompasses methods for treating disorders associated with aberrant expression or activity of a protein of the invention (i.e., Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83). Thus, the invention includes methods for treating disorders associated with excessive expression or activity of a protein of the invention. Such methods entail administering a compound which decreases the expression or activity of the protein. The invention also includes methods for treating disorders associated with insufficient expression or activity of a protein of the invention. These methods entail administering a compound which increases the expression or activity of the protein.
The invention also features methods for detecting a protein of the invention (i.e., Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83). Such methods include: obtaining a biological sample; contacting the sample with an antibody that specifically binds the protein under conditions which permit specific binding; and detecting any antibody-protein complexes formed.
In addition, the present invention encompasses methods and compositions for the diagnostic evaluation, typing, and prognosis of disorders associated with inappropriate expression or activity of a protein of the invention. For example, the nucleic acid molecules of the invention can be used as diagnostic hybridization probes to detect, for example, inappropriate expression of a protein of the invention or mutations in the gene encoding a protein of the invention gene. Such methods may be used to classify cells by the level of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 expression.
The invention encompasses methods for diagnosing a disorder associated with aberrant activity of a protein of the invention, the methods including obtaining a biological sample from a patient and measuring the activity of the protein in the biological sample, wherein increased or decreased activity in the biological sample compared to a control indicates that the patient suffers from a disorder associated with aberrant activity of the protein.
The nucleic acid molecules of the invention can be used as primers for diagnostic PCR analysis for the identification of gene mutations, allelic variations and regulatory defects in the Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 gene. The present invention further provides for diagnostic kits for the practice of such methods.
The invention features methods of identifying compounds that modulate the expression or activity of a protein of the invention by assessing the expression or activity of the protein in the presence and absence of a selected compound. A difference in the level of expression or activity of the protein indicates that the selected compound is capable of modulating expression or activity of the protein. Expression can be assessed either at the level of gene expression (e.g., by measuring mRNA) or protein expression by techniques that are well known to skilled artisans. The activity of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 can be assessed functionally.
The preferred methods and materials are described below in examples which are meant to illustrate, not limit, the invention. Skilled artisans will recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.